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Bovine leukemia virus (BLV), an oncogenic complex retrovirus, is homologous to human T-cell leukemia viruses (HTLV-1 and -2) and simian T-cell leukemia virus. Thirty percent of naturally infected cattle present nonmalignant polyclonal B-cell population expansion in the blood (persistent lymphocytosis [PL]), and 1 to 5% of cows develop lymphocytic leukemia and/or generalized B-cell lymphoma in the 5 to 10 years following infection (for reviews, see references 3 and 30). Since the risk of developing leukemia or lymphoma is greater in cattle with PL than in BLV-seropositive cattle with normal hematological parameters, PL is considered a preneoplastic condition (3). BLV inoculation in sheep is a convenient experimental model for studying the physiopathology of BLV infection because BLV-infected sheep present B-cell lymphoma lesions and B-cell leukemia after a shorter latency period and far more frequently than do cattle (17). We and other authors have reported that some infected sheep also develop nonmalignant B-cell lymphocytosis (7, 20, 28, 31).

In cattle, B-cell lymphocytosis results from an increased number of circulating CD5⁺ B lymphocytes (6, 18, 19) associated with a lower but significant increase of the CD5 B-cell population (19), whereas lymphomas appear to arise exclusively from the CD5⁺ B-cell population (6). The provirus has been detected in both CD5⁺ and CD5 B lymphocytes from long-term-infected animals, with a higher load in CD5⁺ B cells (19, 29). In contrast, in sheep, involvement of CD5⁺ B cells at both the PL and the lymphoma stages has not been consistently observed (1, 16, 20).

In mice, the B-lymphocyte population is classically divided into CD5⁺ B-1a, CD5 B-1b, and "conventional" CD5 B-2 lymphocytes. B-1 cells differ from B-2 cells in many properties (for a review, see reference 9). (i) They display high levels of surface immunoglobulin M (IgM) and low levels of IgD, B220 (CD45RA), and CD11b/CD18, a ₂ integrin normally associated with the myelomonocytic lineage. The CD5 marker allows two subpopulations to be distinguished: a CD5⁺ CD11b⁺ IgM^high IgD^low predominant subset named B-1a and a CD5 CD11b⁺ IgM^high IgD^low minor "sister" population named B-1b that appears to be otherwise identical to the B-1a subset. (ii) They are larger than conventional B-2 lymphocytes. (iii) They comprise a high number of natural polyspecific-antibody-producing cells and their primary immune repertoire is made around the neonatal period. (iv) They present a capacity for self-renewal. (v) Finally, adoptive transfer of B-1 and B-2 cells strongly supports the hypothesis that they belong to separate lineages, although some reports emphasize that the CD5⁺ phenotype can be acquired by "conventional" B2 lymphocytes after stimulation through B-cell activation signals (4). Human homologs of murine B-1 cells, CD5⁺ and/or CD11b⁺ B cells, have also been described in detail (9, 13). Importantly, murine and human B-1 lymphocytes contribute to several pathological disorders, such as autoimmune diseases (9, 21), B-cell malignancies in mice (9), B-cell lymphocytosis associated with human immunodeficiency virus infection (15), and B-cell chronic lymphocytic leukemia in humans (10).

Our group (31) and Dequiedt et al. (7) recently reported that BLV protects sheep peripheral blood mononuclear cells (PBMCs) from spontaneous ex vivo apoptosis. Both reports also indicated that BLV promotes the survival of infected B lymphocytes. These findings suggested that BLV-induced increased survival in B cells is involved in the development of lymphocytosis.

In this study, we first show that BLV infection in sheep induces PL that results from the accumulation of B cells carrying the CD11b/CD18 integrin with variable coexpression of the CD5 molecule, whereas the CD11b B-cell population does not significantly expand. We then further describe the interactions of BLV with the sheep CD11b⁺ B (B-1-like)-cell population. Altogether our data indicate that although both sheep B-1- and B-2-like cells become infected and can express BLV, only B-1 cells show a susceptibility to BLV-associated lymphocytosis and increased survival. Our result also further support the involvement of increased cell survival in the development of lymphocytosis.

B-cell lymphocytosis in BLV-infected sheep mainly results from the expansion of the CD11b⁺ B (B-1-like)-lymphocyte subset. As CD5 and CD11b have been detected on B lymphocytes from lymphocytotic cows (18), we analyzed the expression of these markers on blood B cells from sheep infected for 4 to 6 years with BLV. When the experiments were performed, two-thirds of the infected sheep had high levels of PL on the basis of their increased B-cell/T-cell ratio and their absolute B-cell numbers (Table 1).

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Expansion of the CD5+ and CD11b+ B-cell subsets in BLV-infected sheep. (A) The CD5 and CD21 markers were detected on PBMCs from a control sheep and a BLV-infected sheep with PL by incubating the cells with MAbs CC17 and DU2-104 followed by FITC-conjugated F(ab')2 goat anti-mouse IgG1 (Caltag Laboratories, San Francisco, Calif.) and a phycoerythrin-conjugated F(ab')2 goat anti-mouse IgM antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.). The percentages of cells in the different subsets are indicated. (B) Double labeling for CD11b (MAb CC125) and CD21 (MAb DU2-104) detection on PBMCs from the same sheep as for panel A. The percentages of cells in the different subsets are indicated.

BLV viral loads are similar in B-1- and B-2-like cell subsets from newly infected sheep, and the virus accumulates in B-1-like cells from long-term-infected sheep. The expansion of the B-1-like cell subset could be the result of a specific propensity of this subset to become infected by replicating BLVs. In order to determine whether this is the case, we looked at the viral loads in B-1- and B-2-like cell subsets from newly infected sheep before the onset of lymphocytosis. Previous reports had indicated that the initial viral replication, around 3 to 4 weeks after infection, was important (12). We thus collected PBMCs from 4-week-infected young sheep, doubly labeled them for CD21 and CD11b detection, and sorted the CD11b⁺ and CD11b B cells using flow cytometry. The contamination with cells originating from the theoretically excluded B-cell subset was below 4%. The sorted populations (5 × 10⁴ cells) were lysed and subjected to two independent PCRs, one for BLV detection (forward primer [position 4761], 5'CGCTCTCCTGGCTACTGACC3'; reverse primer [position 5184], 5'ACCGATCTGCCCCCACATAAG3') and the other for the detection of the endogenous glyceraldehyde-3-phospho-dehydrogenase (G3PDH) gene (forward primer, 5'GACCCCTTCATTGACCTCAACTACA3'; reverse primer, 5'CATGTGGGCCATGAGGTCCACCAC3'). Twenty-seven cycles were performed for both reactions as follows: 45 s at 91°C, 45 s at 58°C, and 45 s at 71°C. The PCR products were transferred onto a nylon membrane, revealed with ³²P-labeled probes, and quantitated with the PhosphorImager system (Molecular Dynamics, Sunnyvale, Calif.). The PCRs were performed under strictly defined conditions such that a linear relationship between the initial cell number and the PCR signal intensity was obtained. In order to determine the limit of detection for BLV provirus, we included PCRs with linearized plasmid pBLV13 serially diluted in an uninfected PBMC lysate. The detection limit under our PCR conditions corresponded to 0.5% of cells infected with one viral copy per sample. The intensity of the BLV signal obtained in the two B-cell populations indicated that at most 2.5% of the B cells were infected at that time. Under such conditions, the theoretical signal corresponding to 0.1% of contaminating infected B cells is below the detection level. The BLV signal was normalized between samples by using the G3PDH internal control (Fig. 2A), and a viral load ratio (VLR) for the CD11b⁺ B cells and the CD11b B cells was calculated (Fig. 2B). In sheep 27, the VLR was 1.7 ± 0.17 (mean ± standard error of the mean [SEM] from eight independent PCRs), and in sheep 42, the VLR was 1.2 ± 0.2 (n = 8) (Fig. 2B). At 4 weeks after infection, the CD11b⁺ B-cell number showed 2- and 1.2-fold increases over the values for the day of infection in sheep 27 and sheep 42, respectively, whereas the CD11b B-cell number was unchanged; this increase probably accounts for the slightly higher viral load detected in the CD11b⁺ B cells. Overall, these results indicate that there is no restricted or clear preferential viral tropism for the CD11b⁺ B-cell population compared to the CD11b B-cell population at the beginning of infection.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   BLV viral loads in CD11b+ and CD11b B cells in newly infected and long-term-infected sheep. (A) BLV provirus detection in sorted (by fluorescence-activated cell sorting) CD11b+ B and CD11b B cells from two 4-week-infected sheep (sheep 27 and 42). Uncultured CD11b+ and CD11b B cells were sorted, lysed, and analyzed for the detection of BLV provirus and the endogenous G3PDH gene by PCR, followed by Southern blotting and 32P-specific probing. The signals were analyzed with a PhosphorImager. The BGR obtained for each sorted population is shown. (B) VLRs for the BGRs of the CD11b+ and CD11b B-cell subsets were calculated for newly infected (4-week-infected) sheep and long-term (6-year)-infected sheep. Means and SEMs of the VLRs obtained for 8 (sheep 27 and 42) and 12 (sheep 105, 79, and 121) independent PCR experiments are shown.

BLV-induced protection from apoptosis in B cells mainly affects the CD11b⁺ B-cell subset. B lymphocytes from BLV-infected sheep were shown to be resistant to spontaneous apoptosis, in contrast to B lymphocytes from uninfected sheep (7, 31). As CD11b⁺ B lymphocytes are the major contributors to PL, the BLV-induced increase in cell survival was analyzed in CD11b⁺ and CD11b B lymphocytes. PBMCs were cultured for 48 h and then triply labeled with an anti-CD21 MAb, an anti-CD11b MAb, and fluorescein isothiocyanate (FITC)-conjugated annexin V (Boehringer Mannheim, Mannheim, Germany); the percentages of surviving (i.e., annexin V negative) among CD21⁺ CD11b⁺ and CD21⁺ CD11b lymphocytes could thus be established (Fig. 3). As shown in Fig. 3 and Table 3, CD11b⁺ B lymphocytes from BLV-infected sheep presented a marked increase in survival compared to controls. Conversely, the ex vivo survival of the CD11b B lymphocytes appeared to be only slightly increased. The data also indicate that the magnitude of the ex vivo survival in B-1-like cells increases with the clinical stage (Table 3).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   CD11b+ B cells from BLV-infected sheep show increased ex vivo survival. PBMCs from control and BLV-infected sheep with low- and high-level PL were cultured for 48 h and labeled with FITC-annexin V and for detection of the CD11b and the CD21 markers [CC125 followed by a tricolor conjugated F(ab')2 goat anti-mouse IgG1 antibody (Caltag Laboratories) and DU2-104 followed by a phycoerythrin-conjugated F(ab')2 goat anti-mouse IgM antibody, respectively). The cells positive for both CD11b and CD21 (CD11b+) were gated, as were the cells positive for CD21 and negative for CD11b (CD11b). The proportions of surviving cells (annexin V negative) among the gated B cells are indicated.

View this table: [in this window] [in a new window]   TABLE 3.   Survival of BLV-infected CD11b+ and CD11b B cells

BLV-induced protection from apoptosis is not related to restricted viral expression in the CD11b⁺ B-cell subset. We (31) and Dequiedt et al. (7) showed previously that the majority of BLV-expressing cells ex vivo were resistant to apoptosis, suggesting a direct role for the virus in the increased lymphocyte life span. The resistance to apoptosis limited to the B-1-like cells that we saw here could have been the result of restricted viral expression in this subpopulation. We thus analyzed viral expression in both CD11b⁺ and CD11b B cells ex vivo. PBMCs from sheep 79 cultured for 48 h were labeled for detection of the CD11b and CD21 markers, fixed, permeabilized in 70% methanol, and analyzed for expression of the BLV major capsid protein, p24, by using a pool of anti-BLV p24 MAbs (31). By flow cytometry, the CD11b⁺ B cells and the CD11b B cells were gated and were analyzed. Both subsets were found to express p24 (Fig. 4); a higher percentage of CD11b⁺ B cells (46%) than CD11b B cells (18%) expressed p24, reflecting the higher proviral load in the CD11b⁺ B-cell population. Overall, these results demonstrate that the resistance to apoptosis in CD11b⁺ B cells is not associated with a restriction of the viral expression in this cell subset.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   In situ detection of BLV p24 expression in CD11b+ and CD11b B cells. Forty-eight-hour-cultured PBMCs from sheep 79 were labeled with an anti-CD21 MAb (DU2-104 followed by phycoerythrin-conjugated anti-mouse IgM) and an anti-CD11b MAb (ILA-130 followed by FITC-conjugated anti-mouse IgG2a), permeabilized with 70% methanol, and processed for BLV p24 detection (anti-p24 MAbs followed by triply conjugated anti-mouse IgG1). The p24-positive cells among gated CD11b+ and CD11b B cells are shown. The overlaid left histogram represents background fluorescence of permeabilized CD11b+ and CD11b B cells obtained with a control mouse IgG1 antibody to mutant p53 (Ctrl).

Resistance to apoptosis in B-1-like lymphocytes is acquired in vivo. Whereas BLV expression is largely repressed in vivo, a short culturing leads to rapid activation of viral expression (25). The resistance to apoptosis seen in our assay could thus be the result of the high expression of a viral protein that rarely occurs in vivo and/or occurs in small amounts. In order to demonstrate that the activation of viral expression is not required for increased survival in B-1 cells, inhibitors of BLV activation were used in the ex vivo culture. Inhibitors of protein kinase C, such as H7 {[1-(5-isoquinolinylsulfonyl)-3-methylpiperazine dihydrochloride]}, have been previously illustrated to strongly affect the activation of BLV expression (14). We thus treated the cultured PBMCs with H7 (10 and 15 µM; Sigma, St. Louis, Mo.) for 15 h. The time of the culture needed to be shortened to 15 h because H7 induced cell toxicity in the control PBMCs after 48 h of incubation. After 15 h of culturing, the cells from control sheep 190 presented a low level of survival that was not significantly altered by H7 treatments (Fig. 5A). Treatment of sheep 121 PBMCs with H7 was associated with a reduced level of p24 expression at an H7 concentration of 10 µM and with the inability to detect p24 at 15 µM (Fig. 5C). In parallel, H7 did not significantly affect the high survival level of CD11b⁺ B cells (Fig. 5A). A general inhibitor of protein synthesis, cycloheximide (CHX), was used in order to confirm the results obtained with H7. After 15 h, CHX (Sigma) at 10 and 15 µg/ml slightly but not significantly increased the survival of CD11b⁺ B cells from the control sheep (Fig. 5B). CHX treatments of sheep 121 PBMCs totally prevented p24 expression as determined by Western blot analysis (Fig. 5C) and fluorescence-activated cell sorter analyses (data not shown). CHX treatments did not modify the survival of CD11b⁺ B cells from sheep 121. 

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Inhibition of viral activation with H7 or CHX does not alter the increased survival in CD11b+ B cells from BLV-infected sheep with PL. PBMCs from a control sheep (sheep 190) and from a BLV-infected sheep with high-level PL (sheep 121) were cultured for 15 h without or with H7 (A) or with CHX (B). At the end of the culturing, the cells were labeled with FITC-annexin V and for detection of the CD11b (CC125) and CD21 (DU2-104) markers. The percentages of cell survival in each cell subset were obtained for three independent cultures, and means and standard deviations are reported. (C) Inhibition of viral capsid p24 expression with H7 and CHX treatments was analyzed by Western blotting with a pool of anti-BLV p24 MAbs (5 µg/ml) and an antiactin MAb (0.5 µg/ml, clone AC-15; Sigma) as an internal control.

Significance of susceptibility and resistance of B-1-like cells to apoptosis in BLV pathogenesis. B-1 cells in mice and in humans are often present during the development of B-cell leukemia and lymphomas (9), suggesting that these cells are particularly prone to cellular transformation. In the present study, we show that BLV-induced B-cell lymphocytosis in sheep mainly involves a CD11b⁺ B-cell subpopulation that presents phenotypic similarities to murine B-1 cells. In addition, BLV infection confers a potent resistance to spontaneous apoptosis on B-1-like cells and barely affects B-2-like cells, although both subsets are initially infected and both subsets support viral replication. Collectively, our results suggest that the reactivity of B-1-like cells is not due to restricted viral expression in this subset; rather, B-1-like cells may present a peculiar sensitivity to viral products or to cellular gene activation induced by BLV infection.