Lymphocyte Response to Genetically Engineered Bovine Leukemia Virus Proteins in Persistently Lymphocytic Cattle from Israel and the U.S.
Authors: Splitter Gary , Trainin Zeev , Brenner Yacov 
Published: December 2021

The goal of this proposal was to identify proteins of BLV recognized by lymphocyte subpopulations and determine the contribution of these proteins to viral pathogenesis. Our hypothesis was that BLV pathogenesis is governed by the T-cell response and that the immune system likely plays an important role in controlling the utcome of infection. Our studies presented in ths final report demonstrate that T cell competency declines with advancing stages of infection. Dramatic differences were observed in lymphocyte proliferation to recombinant proteins encoded by BLV gag (p12, p15, and p24) and env (gp30 and gp15) genes in different disease stages. Because retroviruses are known to mutate frequently, examinatin of infected cattle from both Israel and the United States will likely detect variability in the immune response. This combined research approach provides the first opportunity to selectively address the importance of T-cell proliferation to BLV proteins and cytokines produced during different stages of BLV infection. Lack of this information regarding BLV infection has hindered understanding lympocyte regulation of BLV pathogenesis. We have developed the essential reagents necessary to determine the prominence of different lymphocyte subpopulations and cytokines produced during the different disease stages within the natural host. We found that type 1 cytokines (IL-2 and IFN-g) increased in PBMCs from animals in early disease, and decreasd in PBMCs from animals in late disease stages of BLV infection, while IL-10, increased with disease progression. Recently, a dichotomy between IL-12 and IL-10 has emerged in regards to progression of a variety of diseases. IL-12 activates type 1 cytokine production and has an antagonistic effect on type 2 cytokines. Here, using quantitative competitive PCR, we show that peripheral blood mononuclear cells from bovine leukemia virus infected animals in the alymphocytotic disease stage express increased amount of IL-12 p40 mRNA. In contrast, IL-12 p40 mRNA expression by PL animals was significantly decreased compared to normal and alymphocytotic animals. To examine the functions of these cytokines on BLV expression, BLV tax and pol mRNA expression and p24 protein production were quantified by competitive PCR, and by immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA expression by BLV-infected PBMCs. In addition, we determined that macrophages secret soluble factor(s) that activate BLV expression, and that secretion of the soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA, and p24 protein production. These findings suggest that macrophages have a key role in regulating BLV expression, and IL-10 produced by BLV-infected animals in late disease stages may serve to control BLV expression, while IL-2 in the early stage of disease may activate BLV expression. PGE2 is an important immune regulator produced only by macrophages, and is known to facilitate HIV replication. We hypothesized that PGE2 may regulate BLV expression. Here, we show that cyclooxygenase-2 (COX-2) mRNA expression was decreased in PBMCs treated with IL-10, while IL-2 enhanced COX-2 mRNA expression. In contrast, addition of PGE2 stimulated BLV tax and pol mRNA expression. In addition, the specific COX-2 inhibitor, NS-398, inhibited BLV expression, while addition of PGE2 increased BLV tax expression regardless of NS-398. These findings suggest that macrophage derived cyclooxygenase -2 products, such as PGE2, may regulate virus expression and disease rogression in BLV infection, and that cytokines (IL-2 and IL-10) may regulate BLV expression through PGE2 production.