Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle

Bovine leukemia virus (BLV) infection causes a significant polyclonal expansion of CD5(+), IgM+ B lymphocytes known as persistent lymphocytosis (PL) in approximately 30% of infected cattle. There is evidence that this expanded B cell population has altered signaling, and resistance to apoptosis has been proposed as one mechanism of B cell expansion. In human and murine B cells, antigen binding initiates movement of the B cell receptor (BCR) into membrane microdomains enriched in sphingolipids and cholesterol, termed lipid rafts. Lipid rafts include members of the Src-family kinases and exclude certain phosphatases. Inclusion of the BCR into lipid rafts plays an important role in regulation of early signaling events and subsequent antigen internalization. Viral proteins may also influence signaling events in lipid rafts. Here we demonstrate that the largely CD5(+) B cell population in PL cattle has different mobilization and internalization of the BCR when compared to the largely CD5-negative B cells in BLV-negative cattle. Unlike B cells from BLV-negative cattle, the BCR in B cells of BLV-infected, PL cattle resists movement into lipid rafts upon stimulation and is only weakly internalized. Expression of viral proteins as determined by detection of the BLV transmembrane (TM) envelope glycoprotein gp30 did not alter these events in cells from PL cattle. This exclusion of the BCR from lipid rafts may, in part, explain signaling differences seen between B cells of BLV-infected, PL, and BLV-negative cattle and the resistance to apoptosis speculated to contribute to persistent lymphocytosis.     

Imbalance of tumor necrosis factor receptors during progression in bovine leukemia virus infection

Previously, we found an up-regulation of tumor necrosis factor alpha (TNF)-alpha and an imbalance of TNF receptors in sheep experimentally infected with bovine leukemia virus (BLV). In order to investigate the different TNF-alpha-induced responses, in this study we examined the TNF-alpha-induced proliferative response and the expression levels of two distinct TNF receptors on peripheral blood mononuclear cells (PBMC) derived from BLV-uninfected cattle and BLV-infected cattle that were aleukemic (AL) or had persistent lymphocytosis (PL). The proliferative response of PBMC isolated from those cattle with PL in the presence of recombinant bovine TNF-alpha (rTNF-alpha) was significantly higher than those from AL cattle and uninfected cattle and the cells from PL cattle expressed significantly higher mRNA levels of TNF receptor type II (TNF-RII) than those from AL and BLV-uninfected cattle. No difference was found in TNF-RI mRNA levels. Most cells expressing TNF-RII in PL cattle were CD5+ or sIgM+ cells and these cells showed resistance to TNF-alpha-induced apoptosis. Additionally, there were significant positive correlations between the changes in provirus load and TNF-RII mRNA levels, and TNF-alpha-induced proliferation and TNF-RII mRNA levels. These data suggest that imbalance in the expression of TNF receptors could at least in part contribute to the progression of lymphocytosis in BLV infection.     

Cellular basis of persistent lymphocytosis in cattle infected with bovine leukemia virus.

Peripheral blood lymphocytes from 14 cattle infected with the bovine leukemia virus (BLV) and 14 BLV-free cattle were examined by the membrane immunofluorescent antibody technique to detect surface immunoglobulin (S-Ig) and by the erythrocyte-antibody-complement (EAC) rosette test for the detection of complement receptors. Direct comparisons of the percentages of S-Ig-bearing cells and EAC rosette-forming cells in both infected and BLV-free animals showed no evidence for the presence of a substantial population bearing one surface marker but not the other. The data showed that cells with surface markers characteristic of B lymphocytes are responsible for most of the increase in peripheral blood lymphocytes which may accompany BLV infection. The release of infectious BLV and the spontaneous uptake of thymidine by short-term cultured peripheral blood lymphocytes from BLV-infected cattle were also studied. The results indicate that both of these activities are function of B lymphocytes.     

Detection of bovine leukemia virus in cattle by the polymerase chain reaction.

Bovine leukemia virus (BLV) is widely distributed in U.S. cattle herds. It infects B lymphocytes and causes neoplastic disease in 5-10% of infected animals. Direct economic losses are incurred as a result of death, reduced milk production and condemnation at slaughter. Thus the identification of cattle infected with BLV is of significant concern to the U.S. cattle industry. For this reason, polymerase chain reaction (PCR) amplification was used to examine seropositive and seronegative cattle for the presence of BLV DNA in peripheral blood mononuclear cells. Using an amplification protocol able to detect 1 viral genome in 100,000 cells, BLV was not detected in 7 seronegative cattle in an infected herd. BLV sequences were detected in 13 of 18 seropositive animals with various levels of infection as determined by in vitro lymphocyte culture and electron microscopy. An active infection was demonstrated in one animal, based on the presence of viral RNA. These findings indicate that PCR is a sensitive method for the detection of BLV in cattle and provides new information regarding the dynamics of the infection.     

Evidence that the spontaneous blastogenesis of lymphocytes from bovine leukemia virus-infected cattle is viral antigen specific.

Cattle lymphocytes cultured for 3 days were found to spontaneously incorporate thymidine (3STI). Under optimal conditions of culture, the median magnitude of 3STI activity in lymphocytes from bovine leukemia virus (BLV)-infected cattle was higher than that of BLV-free cattle, but the ranges of the values overlapped. However, the 3STI activity of most BLV-infected cattle was specifically inhibited by serum containing BLV antibodies, whereas the 3STI activity of BLV-free cattle was not. The 3STI inhibitor copurified with immunoglobulin, and its activity could be absorbed with BLV. Rabbit anti-BLV serum inhibited 3STI, but rabbit anti-BLV p25 did not. These results indicate that BLV infection induces or expands a BLV-specific lymphocyte population. Spontaneous blastogenesis may be indicative of an immune response which controls virus spread.     

In vitro susceptibility of Thai seronegative donor CD8+ T lymphocytes to human immunodeficiency virus-1 (HIV-1) infection

To determine whether CD8+ T lymphocytes from Thai donor cells are susceptible to HIV-1 infection, undepleted peripheral blood mononuclear cells (PBMC) and CD8-enriched PBMC were infected with HIV-1 Thai subtype B and CRF01_AE (E) primary isolates. Virus kinetics in HIV-1 infection of CD4+ and CD8+ T lymphocytes peaked at day 7 or 10 post infection (pi); the TCID50 used for cell infection was proportional to the level of p24 production in the cultures. We also found that the level of p24 antigen in the supernatants of infected undepleted PBMC was significantly higher than that of infected CD8-enriched PBMC. Interestingly, both single positive T lymphocytes (CD4+ and CD8+ T lymphocytes) as well as double positive CD4+/CD8+ T lymphocytes were infected with HIV-1. The double positive T lymphocytes in PBMC were found only in the presence of both CD4+ and CD8+ T lymphocytes. The majority of p24+/CD4-/CD8- T lymphocytes were HIV-1 infected CD4 down-modulated PBMC. This report provides direct evidence that single positive CD8+ T lymphocytes and double positive CD4+/ CD8+ T lymphocytes from Thai donors can be infected with HIV-1 subtypes B and E in vitro.     

Antiviral activity of shiga toxin 1: suppression of bovine leukemia virus-related spontaneous lymphocyte proliferation

Human infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemorrhagic colitis. The Stxs belong to a large family of ribosome-inactivating proteins (RIPs) that are found in a variety of higher plants and some bacteria. Many RIPs have potent antiviral activity for the plants that synthesize them. STEC strains, both virulent and nonvirulent to humans, are frequently isolated from healthy cattle. Interestingly, despite intensive investigations, it is not known why cattle carry STEC. We tested the hypothesis that Stx has antiviral properties for bovine viruses by assessing the impact of Stx type 1 (Stx1) on bovine peripheral blood mononuclear cells (PBMC) from cows infected with bovine leukemia virus (BLV). PBMC from BLV-positive animals invariably displayed spontaneous lymphocyte proliferation (SLP) in vitro. Stx1 or the toxin A subunit (Stx1A) strongly inhibited SLP. Toxin only weakly reduced the pokeweed mitogen- or interleukin-2-induced proliferation of PBMC from normal (BLV-negative) cows and had no effect on concanavalin A-induced proliferation. The toxin activity in PBMC from BLV-positive cattle was selective for viral SLP and did not abrogate cell response to pokeweed mitogen- or interleukin-2-induced proliferation. Antibody to virus or Stx1A was most effective at inhibiting SLP if administered at the start of cell culture, indicating that both reagents likely interfere with BLV-dependent initiation of SLP. Stx1A inhibited expression of BLV p24 protein by PBMC. A well-defined mutant Stx1A (E167D) that has decreased catalytic activity was not effective at inhibiting SLP, suggesting the inhibition of protein synthesis is likely the mechanism of toxin antiviral activity. Our data suggest that Stx has potent antiviral activity and may serve an important role in BLV-infected cattle by inhibiting BLV replication and thus slowing the progression of infection to its malignant end stage.     

Transformation studies with a human T-cell leukemia virus type 1 molecular clone

In in vitro studies human T-cell leukemia virus type 1 (HTLV-1) may be produced by stable or transient transfection of target cells with an infectious molecular clone. Studies using primary human T cells, the natural targets of HTLV-1 infection, are hampered by difficulty in achieving significant infection with cell-free virus and a poor efficiency of transfection of primary cells. A method is described for the generation of stable cell lines expressing HTLV-1 from an infectious proviral clone. The stably transfected cells can be irradiated and cocultured with human peripheral blood mononuclear cells (PBMC) resulting in infected primary cells. These cells become immortalized, IL-2 dependent lines, which contain integrated copies of provirus and express a full spectrum of viral proteins. Analysis of cellular markers indicates that immortalized cell lines consist of CD3+/CD4+ T cells, matching the most common adult T-cell leukemia (ATL) cell phenotype. The method described has great utility in the study of the replication and transformation capacity of HTLV and HTLV mutant viruses in their natural targets, primary human T lymphocytes.     

Soluble interleukin 2 receptor release, interleukin 2 production, and interleukin 2 receptor expression in activated T-lymphocytes in vitro.

Following activation in vitro, peripheral blood mononuclear cells (PBMC) express cell-associated interleukin-2 receptors (IL2R). The present study was undertaken to define the proportion of T lymphocyte subsets that express the IL2R (CD25 antigen) upon different mitogenic stimulation. Double immunofluorescence staining with different fluorochromes, fluorescein isothiocyanate and phycoethyrin, was applied for identification of IL2R positive cells and individual lymphocyte subset. The exact percentage of individual activated lymphocyte subset bearing IL2R was enumerated by photographic counting. There was paucity of IL2R in freshly isolated, unstimulated peripheral blood, PBMC cultured without mitogen, and cultured B lymphocytes. Following pokeweed mitogen stimulation in vitro, 19% of CD4 (T-helper/inducer) lymphocytes and 14% of CD8 (T-suppressor/cytotoxic) lymphocytes expressed IL2R. Similarly, 25% of CD4 lymphocytes and 19% of CD8 lymphocytes expressed IL2R following phytohemagglutinin stimulation in vitro. Contrary to the reported data of Tac-positive cells in human lymphoid tissues, our study revealed that, upon lectin mitogen stimulation, approximately 55% of IL2R positive PBMC were CD4 lymphocytes, and 45% of them were CD8 lymphocytes. These observations imply the plausible notion that interleukin-2 mediated immune activation of T lymphocytes in PBMC is different from that in local lymphoid organs. It was also demonstrated that the release of soluble IL2R (sIL2R) and IL2 production in supernatant from cultured PBMC varied with different lectin stimulation. A significant correlation was demonstrated between the cellular and soluble IL2R but the production of IL2 from activated mononuclear cells bore no good correlation with either the cellular IL2R expression or the release of sIL2R.     

Specific CD8+ T‐lymphocytes control dissemination of measles virus

Measles continues to be an important cause of childhood mortality in developing countries. Measles virus (MV) is lymphotropic and infects high percentages of B‐ and T‐lymphocytes in lymphoid tissues. Cellular immunity is considered crucial for viral clearance; however, MV‐specific T‐lymphocytes generated during primary infection also constitute a potential target for MV infection. We therefore aimed to identify T‐lymphocyte subsets that can clear MV infection without becoming infected. To this end, we infected human EBV transformed B‐lymphoblastic cell lines (B‐LCL) with a recombinant MV strain expressing enhanced GFP, and co‐cultured these with non‐infected B‐LCL resulting in rapid viral spread. MV‐specific CD8⁺ T‐cell clones efficiently suppressed MV dissemination in autologous and HLA‐matched, but not in HLA‐mismatched B‐LCL. In contrast, CD4⁺ T‐cell clones could not control MV dissemination but became a target for MV infection themselves. Furthermore, PBMC collected 6–9 months after acute measles and stimulated with autologous MV‐infected B‐LCL also efficiently suppressed MV dissemination; this was mediated by the fraction containing CD8⁺ T‐lymphocytes. In conclusion, we have developed a powerful tool to study cellular immunity against measles, and demonstrate that control of MV dissemination is mediated by virus‐specific CD8⁺ rather than by CD4⁺ T‐lymphocytes.     

Effect of platelet-derived factor on expression of bovine leukemia virus genome

Summary Plasma of cattle infected with bovine leukemia virus (BLV) contains a factor, plasma blocking factor (PBF), that inhibits the expression of viral genome in cultured lymphocytes from BLV infected cattle. When platelet lysate was added to this culture, BLV antigen became detectable in the culture and there are some factors (PDF) in platelet lysate which have inhibitory activity against PBF. The PDF was present in platelet from BLV-free cattle as well as BLV-infected cattle at relatively high titer. The effect of platelet lysate against PBF on the expression of BLV genome seemed to be irreversible.     

Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.     

Increased serum neopterin in patients with HIV-1 infection is correlated with reduced in vitro interleukin-2 production.

Recently we have observed that the CD4+ T cell response of peripheral blood mononuclear cells (PBMC) to soluble antigens is the first to be lost in the course of HIV-1 infection followed by the loss of response to HLA alloantigens. In this study we compared serum neopterin concentrations of individuals with early stages of HIV-1 infection (stages WR1 and WR2, Walter Reed staging system) with in vitro interleukin-2 (IL-2) production of PBMC in response to stimulation with soluble antigens (influenza A virus and tetanus toxoid) and alloantigens. Neopterin concentrations were significantly higher in HIV-1-seropositive individuals who showed deficient IL-2 production in response to recall antigens only or to all of the stimuli tested in vitro, compared with HIV-1-seropositive individuals who exhibited no CD4+ T cell defects. No difference in serum neopterin concentrations was observed between the group that was functionally deficient to soluble antigens only versus those who were unresponsive to both types of stimuli. It appears that the selective loss of the MHC self-restricted CD4+ T cell function is associated with an increase in serum neopterin levels. Neopterin concentrations are an estimate of the activation status of macrophages. We conclude that defective in vitro production of lymphokines by T lymphocytes is associated with activated macrophages in vivo.     

Properties of density gradient-fractionated peripheral blood leukocytes from cattle infected with bovine leukemia virus.

Discontinuous bovine serum albumin gradients were used to fractionate peripheral blood leukocytes from bovine leukemia virus (BLV)-free and BLV-infected cows. The release of infectious BLV and spontaneous incorporation of [3H]thymidine were not properties of density gradient-fractionated leukocytes from a BLV-free cow. When leukocytes from BLV-infected cattle were fractionated, B lymphocytes which spontaneously incorporated [3H]thymidine could be separated as a distinct subpopulation from B lymphocytes which replicated infectious BLV. Density gradient fractionation of leukocytes from a cow with lymphosarcoma is also reported. A fall in lymphocyte count at the time of tumor development is attributed to the loss of B lymphocytes which spontaneously incorporate [3H]thymidine.     

Bovine leukemia virus gp30 transmembrane (TM) protein is not tyrosine phosphorylated: examining potential interactions with host tyrosine-mediated signaling

Bovine leukemia virus (BLV) causes persistent lymphocytosis, a preneoplastic, polyclonal expansion of B lymphocytes. The expansion increases viral transmission to new hosts, but the mechanisms of this expansion have not been determined. We hypothesized that BLV infection contributes to B-cell expansion by signaling initiated via viral transmembrane protein motifs undergoing tyrosine phosphorylation. Viral mimicry of host cell proteins is a well-demonstrated mechanism by which viruses may increase propagation or decrease recognition by the host immune system. The cytoplasmic tail of BLV transmembrane protein gp30 (TM) has multiple areas of homology to motifs of host cell signaling proteins, including two immunoreceptor tyrosine-based activation motifs (ITAMs) and two immunoreceptor tyrosine-based inhibition motifs (ITIMs), which are homologous to B-cell receptor and inhibitory co-receptor motifs. Signaling by these motifs in B cells typically relies on tyrosine phosphorylation, followed by interactions with Src-homology-2 (SH2) domains of nonreceptor protein tyrosine kinases or phosphatases. Phosphorylation of tyrosine residues in the cytoplasmic tail of TM was tested in four systems including ex vivo cultured peripheral blood mononuclear cells from BLV infected cows, BLV-expressing fetal lamb kidney cell and bat lung cell lines, and DT40 B cells transfected with a fusion of mouse extracellular CD8 and cytoplasmic TM. No phosphorylation of TM was detected in our experiments in any of the cell types utilized, or with various stimulation methods. Detection was attempted by immunoblotting for phosphotyrosines, or by metabolic labeling of cells. Thus BLV TM is not likely to modify host signal pathways through interactions between phosphorylated tyrosines of the ITAM or ITIM motifs and host-cell tyrosine kinases or phosphatases.     

T cell immunophenotypes and DR antigen expression in intravenous drug users. Relationship to human immunodeficiency virus serology

Thirty-six intravenous drug users were studied for peripheral blood mononuclear cell (PBMC) immunophenotypes and human immunodeficiency virus (HIV) serological profiles. This population has a high risk for developing HIV infection. Half (18/36) were HIV antibody (Ab) negative (-) and half were positive (+). Total T lymphocytes (CD3+ and CD2+) were not different between HIV Ab-negative and HIV-positive groups. Unactivated T(CD3+DR-) cells/mm3 were less (p = 0.003) in HIV Ab-positive patients (1,467 +/- 628) compared to HIV Ab-negative patients (2,190 +/- 695). T-helper (CD4+) cells/mm3 were also less in HIV Ab-positive patients (762 +/- 344 vs. 1,161 +/- 419, p = 0.005). The most significant difference was in activated T lymphocyte CD3+DR+) percentages where the mean was 9.6% in those HIV Ab-positive compared to 3.8% in seronegatives (p less than 0.001). Preliminary studies showed that in vitro naloxone treatment of PBMC had no effects on immunophenotypic expression except for CD3+DR+ lymphocytes, where a significant reduction was observed in the HIV Ab-positive group (p = 0.022) but not in the HIV ab-negative group. These findings suggest that in certain populations, activated T cells may be an early manifestation of HIV infection.