FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/ macrophages

doi:10.1016/0165-2427(91)90102-I

Veterinary Immunology and Immunopathology

Volume 27, Issues 1–3, January 1991, Pages 207-213

https://doi.org/10.1016/0165-2427(91)90102-I Get rights and content

Abstract

The eighteen monoclonal antibodies (mAbs) to B cells and the fourteen mAbs to accessory cells submitted to the workshop were analysed by FACS on three established, bovine leukemia virus (BLV)-infected bovine cell lines. Several mAbs of previously defined specificity were run in parallel. This analysis allowed us to gain further insight on the precise phenotype of those peculiar cells and to cluster the submitted mAbs according to their staining patterns. The BLV-infected cell lines seemed to belong to the B cell type though some of them lack detectable surface immunoglobulins. Moreover, all lines express the CD5 T cell marker and several myeloid markers.

References (17)

A. Depelchin et al.
Bovine leukemia virus (BLV) infected B cells express a marker similar to the CD5 T cell marker
Immunol. Letters
(1989)
C.L. Baldwin et al.
Characterization of a subset of bovine T lymphocytes by mAbs and function: similarity to those defined by human T4 and murine L3T4
J. Immunol.
(1986)
C.L. Baldwin et al.
MAbs which rest with bovine T lymphocytes antigens and induce blastogenesis: tissue distribution and functional characteristics of the target antigens
Immunology
(1988)
J. Braun
Spontaneous in vitro occurrence and long term culture of murine B lymphoblast cell lines
J. Immunol.
(1983)
A. Bruny et al.
Bovine leukemia: facts and hypotheses derived from the study of an infectious cancer
Cancer Survey
(1987)
W.F. Davidson
W.C. Davis et al.
Bovine pan-T cell mAb reactive with a molecule similar to CD2
Immunology
(1988)
A. De la Hera et al.
Coexpression of MAC-1 and P150/95 on CD5 + B cells. Structural and functional characterization in human chronic lymphocytic leukemia
Eur. J. Immunol.
(1988)

There are more references available in the full text version of this article.

Cited by (15)

Translocation of the B cell receptor to lipid rafts is inhibited in B cells from BLV-infected, persistent lymphocytosis cattle
2003, Virology
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.
Bovine leukemia virus gp30 transmembrane (TM) protein is not tyrosine phosphorylated: Examining potential interactions with host tyrosine-mediated signaling
2002, Virus Research
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 CD8α 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.
Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus
1999, Veterinary Immunology and Immunopathology
The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.
Effect of permeabilization on peripheral blood lymphocytes of bovine leukemia virus (BLV)-infected cattle
1997, Veterinary Immunology and Immunopathology
Cell surface proteins serve as markers for immunophenotypic characterization of lymphocyte subsets by appropriate monoclonal antibodies and fluorescence-activated cell sorter (FACS) analysis. By the same method, internal antigens or those that are only partially expressed on the cell surface can be determined after permeabilization of the cells.
Peripheral blood lymphocytes obtained from bovine leukemia virus (BLV)-infected cattle and from BLV-free cattle were permeabilized and several lymphocyte populations were examined.
BoCD4, BoCD8 and three CD4⁻ CD8⁻T-cell subsets retained their original frequencies after permeabilization in both groups of animals. The recognition of the B-B2 lymphocyte molecule was only partially expressed on the cell surface of intact lymphocytes and was further revealed on permeabilization. The frequency of permeabilized, but not intact, cells stained with this mAb was significantly higher for BLV-infected cattle than for BLV-free animals (P = 0.006). Reactivities of an anti-heat shock protein (Hsp) 70 were measured before and after permeabilization of PBLs. Similar increased cell frequencies were obtained for both groups of bovines.
These data indicate that flow cytometry studies should be conducted on both permeabilized and intact cells for a better assessment of protein expression on the cell surface, as well as in the cytoplasm.
Bovine leukemia virus: Early reflections in blood after an experimental infection of calves
1997, Comparative Immunology, Microbiology and Infectious Diseases
In order to study early alterations in the blood following infection with bovine leukemia virus (BLV) in the natural host, 15 calves were inoculated with blood from a BLV-positive donor cow. The humoral immunological response was followed by ELISA for 2 months. Seroconversion to BLV was demonstrated at 4–5 weeks post-infection. Total and differential leukocyte counts were performed. Acute lymphocytosis was observed at the time of seroconversion in the majority of the experimental calves. By the aid of monoclonal antibodies (mAbs), the proportion as well as the total number of lymphoid cells were studied in four of the calves, applying analytical flow cytometry. At the time of seroconversion the percentage of B-cells increased from 19.1 ± 7.5% to 37.9 ± 15.8%, and the T-cells (CD2⁺) decreased from 36.7 ± 7.3% to 22.7 ± 6.0%, the latter being attributable to decreases in the percentage of CD4⁺ as well as CD8⁺ T-cells for the infected calves together. Subsequently, altered $B T$ ratios were observed. In one of the calves an increase in the absolute number of CD5⁺ cells coincided with an increase in total B-cells. The early phenotypic alterations in lymphocyte subsets, before and after seroconversion to BLV, were comparable to those of non-lymphocytotic and persistent lymphocytotic cattle, respectively. Sera from 15 calves were tested for the presence of interferon (IFN), as measured by antiviral activity. BLV does not appear to induce the production of IFN.
Dans le but d'étudier les modifications précoces induites dans le sang après une infection par le virus de la leucose bovine (BLV) chez l'hôte naturel, 15 veaux ont été infectés avec le sang d'une vache donneuse positive pour le BLV. La réponse humorale immunologique a été suívie par un test ELISA pendant deux mois. Quatre à cinq semaines après l'infection, une séroconversion vis à vis du BLV a été mise en évidence. La numération totale et différentielle des leucocytes a été faite. Une lymphocytose aiguë a été observée au moment de la séroconversion chez la majorité des veaux expérimentalement infectés. Avec l'aide des anticorps monoclonaux (mAbs) la proportion ainsi que le nombre total des cellules lymphoïdes ont été étudiés dans quatre des veaux par analyse en cytométrie de flux. Au moment de la séroconversion le pourcentage des cellules B a augmenté de 19,1 ± 7,5% jusqu'à 37,9 ± 15,8% et les cellules T (CD2) ont diminué de 36,7 ± 7,3% jusqu'à 22,7 ± 6,0%, ce dernier changement peut être attribué à la diminution du pourcentage des lymphocytes T CD4 ainsi que les CD8. Les altérations du rapport $B T$ ont été observées. Chez un des veaux, les augmentations du nombre absolu de cellules CD5 ont coïncidé avec une augmentation du nombre de cellules B totales. Les premières altérations phénotypiques dans les sous-populations lymphocytaires, avant et après la séroconversion BLV, ont été comparables à celles connues chez des bovins non-lymphocytosiques ou lymphocytosiques persistants. La présence d'IFN dans le serum a été recherchée chez 15 veaux par mesure de l'activité antivirale. Le BLV ne semble pas provoquer une production de IFN.
B-1a, B-1b and conventional B cell lymphoma from enzootic bovine leukosis
1996, Veterinary Immunology and Immunopathology
In order to characterize the phenotypes of tumor cells and to clarify from which B cell lineage the lymphomas were derived, ten cows with enzootic bovine leukosis were examined by means of immunohistologic staining and flow cytometry. The tumor cells expressed mainly major histocompatibility complex (MHC) class II⁺ ( $1010$ ), BoCD11b⁺ ( $910$ ), IgG₁⁺ ( $810$ ), B-B2⁺ ( $810$ ) BoCD5⁺ ( $710$ ), and λ light chain⁺ ( $710$ ). Tumor cells from only one animal expressed sIgM⁺ ( $110$ ). Tumor cells from all ten animals were negative for IgG₂, BoCD3, BoCD4, BoCD8, WC1-N2, and IL-2Rα. The phenotypes of these tumor cells were all slightly different, suggesting that bovine leukemia virus (BLV)-induced lymphoma expresses phenotypic diversity. Moreover, tumor cells from seven cattle coexpressed BoCD5 and BoCD11b (B-1a cells). On the other hand, tumor cells from two of them only expressed BoCD11b (B-1b cells), and those from one were negative for both BoCD5 and BoCD11b (conventional B cells). Therefore, we concluded that BLV-induced lymphoma cells can be derived from B-1a, B-1b and conventional B cells.

View all citing articles on Scopus

View full text

FACS analysis of bovine leukemia virus (BLV)-infected cell lines with monoclonal antibodies (mAbs) to B cells and to monocytes/ macrophages

Abstract

Immunol. Letters

Characterization of a subset of bovine T lymphocytes by mAbs and function: similarity to those defined by human T4 and murine L3T4

J. Immunol.

MAbs which rest with bovine T lymphocytes antigens and induce blastogenesis: tissue distribution and functional characteristics of the target antigens

Immunology

Spontaneous in vitro occurrence and long term culture of murine B lymphoblast cell lines

J. Immunol.

Bovine leukemia: facts and hypotheses derived from the study of an infectious cancer

Cancer Survey

Bovine pan-T cell mAb reactive with a molecule similar to CD2

Immunology

Coexpression of MAC-1 and P150/95 on CD5 + B cells. Structural and functional characterization in human chronic lymphocytic leukemia

Eur. J. Immunol.